Activation of guanylate cyclase by nitrosyl hemoglobin (NOHb) or activator with MgC1-2 or MnC1-2 was investigated. Maximal enzyme was observed with NOHb and MgC1-2, MnC1-2, and activator or MnC1-2, activator, and NOHb. Excess of Mg-2+ or Mn-2+ above substrate concentration was requried for optimal activity. NOHb and activator both did not alter kinetic constants of MgGTP, linear kinetic relation, or MnGTP, a negative cooperative nature. Activation by both compounds was inhibited by methylene blue and 1,10-phenanthroline. Inactivation of cyclase at 0 C was prevented by Hb, phenyl-Sepharose, methylene blue, or methyl viologen. Three monoclonal antibodies have been studied, only one of which appears to inhibit guanylate cyclase.